The overall goal is to understand the molecular events involved in the tissue-specific, regulated expression of the prolactin gene. The prolactin gene is regulated at the transcriptional level by a number of hormones including estradiol, epidermal growth factor, thyrotropin releasing hormone and dopamine. The proposed studied seek to examine the mechanisms which permit the multihormonal regulation of prolactin transcription. In particular attention will be focused on intracellular regulators of prolactin gene transcription, DNA sequences which are important for prolactin gene transcription and on DNA-binding factors which interact with these sequences. The specific aims are approaches include: 1) The role of the catalytic subunit of the cAMP-dependent protein kinase in mediating cAMP effects on prolactin gene transcription will be examined by using a synthetic gene coding for the heat stable inhibitor of the kinase 2) Structural features of the estrogen response element of the rat prolactin gene which are necessary for binding the estradiol receptor and for stimulation of transcription will be examined through construction of synthetic estrogen response elements and mutagenesis of the response element 3) Features of the DNA binding domain of the estrogen receptor which are important for interaction with the prolactin estrogen response element will be explored through mutagenesis of the cloned receptor cDNA. 4) Linker scanning mutagenesis will be used to dissect the role of specific DNA sequences in the immediate 5' flanking region and an upstream enhancer/estrogen response element of the prolactin gene 5) DNA binding proteins which interact with specific prolactin gene sequences will be identified by mobility shift assay, purified by affinity chromatography and cDNAs cloned. These studies should provide increased understanding of the general mechanisms involved in the regulation of gene expression as well as the specific regulation of this physiologically important hormone.